Human cells contain many copies of a 16,569 base pair mitochondrial genome to permit expression of only 13 proteins that are all essential subunits of complexes required for respiration. These proteins are translated on mitochondrial ribosomes assembled by combining three ribosomal RNAs with about 80 nucleus-encoded proteins imported into mitochondria. These mitochondrial ribosomal proteins (MRPs) have only recently been identified and several of them, including MRPS16, MRPS22, MRPL3 and MRPL44 have been implicated in inherited genetic disorders. Despite its critical importance, the pathway for mitoribosome assembly is virtually unknown, establishing this as a fertile subject for study. Mitochondrial ribosome assembly and function are altered in aminoglycoside-induced deafness and neurodegenerative diseases. Impaired mitochondrial ribosome assembly resulting in mitonuclear protein imbalance may also contribute to the progressive mitochondrial dysfunction observed with aging. We propose to use a novel approach we developed to study mitochondrial ribosome assembly using stable isotope pulse-chase labeling in cell culture (pulse-chase SILAC) and mass spectrometry. Our extensive preliminary results show that certain proteins bind newly- synthesized rRNA at mtDNA nucleoids, possibly while transcription is continuing; these are candidates for early ribosome assembly proteins. Others only join the ribosome later, after it is no longer tightly linked to the nucleoid. We propose to use refined pulse-chase methods to improve understanding of the mechanism, kinetics and efficiency of mitoribosome assembly. Recent cryo-electron microscopy studies have discovered the tRNAvaline as a novel component of the large subunit. We will test a model in which cleavage of the tandemly transcribed 12S rRNA-tRNAval-16S rRNA into three separate RNAs must be coordinated with binding of newly synthesized MRPs. We will determine whether newly-synthesized tRNAval is incorporated into the ribosome along with the tandemly-transcribed 16S rRNA by a transcription-coupled assembly process, or whether a preexisting copy of tRNAval is recruited into the ribosome. We will study how the assembly process is distorted when the system is perturbed by depletion of an individual MRP or of assembly factors that are not themselves ribosomal components. In light of our finding that early assembly takes place at the mtDNA nucleoid, we will extend our efforts to study mitoribosome assembly in cells with disordered nucleoid structure. We hypothesize that if altered nucleoid structure affects rRNA synthesis and processing, this could lead to accumulation of MRPs that cannot participate efficiently in ribosome assembly, leading to a mitochondrial unfolded protein response. Impact: The proposed research will provide mechanistic insight into the process of mitoribosome assembly, clarify its dependence on RNA processing, and investigate the consequences of pathological alterations that alter assembly, including triggering of the unfolded protein response. Long term, this will pave the way for a vastly improved understanding of mitoribosome biogenesis, which is essential in order to understand the pathogenesis of mitochondrial disorders resulting from mitochondrial translation defects.